Interstrand crosslinks (ICLs) are deleterious DNA damages in which both strands are covalently bound together, generating a stalled replication state cytotoxic to cells. Defective repair of ICLs is associated with Fanconi Anemia syndrome (FA) and predisposition to cancer. At least seventeen FA genes have been identified up to date. This proposal focuses on a specific FA protein, FANCA, a member of the FA core complex that is mutated in 64% of the entire FA patient population and does not have a clear function. Our preliminary study shows that FANCA destabilizes (unwinds) DNA helix and recognizes DNA interstrand crosslink damage (ICL) in a replication fork. Intriguingly, FANCA anneals single-strand DNA and catalyzes strand exchange as well. Furthermore, FANCA synergistically interacts with Rad51, the major recombinase in double strand break (DSB) repair. Based on these data, we hypothesize that FANCA directly participates in repair of interstrand crosslinks through its DNA-destabilizing (`unwinding') activity and facilitates subsequent repair of double strand DNA breaks by catalyzing strand annealing and exchange. In order to delineate the role of FANCA in DNA repair, we will use a biochemically defined in vitro system, a cell-based DSB repair study system, and a living cell imaging system to accomplish four aims: 1) Delineate the molecular mechanism of how FANCA catalyzes DNA `unwinding', single-strand annealing, and strand exchange. 2) Determine the role of FANCA in recognition and incision of DNA interstrand crosslinks. 3) Study the biological role of FANCA in double strand break repair. 4) Examine the physiological role of the DNA `unwinding', single- strand annealing, and strand exchange activities of FANCA in the etiology of Fanconi anemia. Understanding the role of FANCA in DNA repair will not only contribute to the overall clarification of the ICL repair process, but also provide novel insights into the etiology of Fanconi anemia and its associated cancer.